Infections with the human pathogenic papilloma virus, which is widely distributed among the population, are among the most common sexually transmitted viral diseases. However, it is also possible for newborns to become infected through the birth channel. The consequences of an HPV disease usually involve harmless dermal symptoms. To date, about a hundred different types of this virus have been identified. Papilloma viruses are classified into cutaneous types, which mainly cause keratinizing lesions in the epithelium, and mucosal types, which in particular affect the mucous membranes. The viruses are also classified further into types associated with benign lesions (low-risk types) and types that are associated with preneoplastic and malignant epithelial changes (high-risk types). Known high-risk types are, for example, types 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, and 82. Known low-risk HPV types are, for example, types 6, 11, 40, 42, 53, 54, 57, and MM8.
About 25 papilloma virus types are causally associated with diseases that occur in the female genital area. HPV infections manifest themselves in the female anogenital area, in particular in the form of condylomatous, dysplastic, and neoplastic lesions. Since 99.7% of all cervical cancers contain papilloma virus DNA, it is now generally accepted that human papilloma viruses are undeniable risk factors for the development of this type of cancer. Epidemiological and molecular studies have shown that continuous infection with high-risk HPV types, in particular HPV types 16 and 18, plays a significant role in the development of cervical carcinoma.
Cervical cancer and other cancer diseases that are epidemiologically associated with papilloma viruses, for example certain forms of skin cancer, are preventable diseases if early detection and treatment are assured. A reliable process for diagnosing the presence of an HPV infection is therefore essential to effective therapy.
In diagnostic terms, it is possible to distinguish between three forms of HPV infections: clinical, subclinical, and latent HPV infections. Epithelial changes that are associated with HPV and that occur in the subclinical or clinically manifest stage of the infection can be detected relatively well using cytological techniques. Early stages of cervical cancer therefore are currently identified mainly by taking a cell smear from the portio or cervix in combination with colposcopy. While cytological methods have contributed to a significant decrease in the incidence of cervical carcinomas in recent years, they do not provide completely satisfactory results. It is not possible to obtain a prognosis on the further development of individual lesions. Moreover, the cytological methods suffer from relatively large subjective errors, and they are not standardized.
Since it is not possible to breed and culture HPV, the detection of HPV in the laboratory is accomplished by identifying viral DNA or shell proteins. For example, group-specific antigens (capsid antigens) of the papilloma viruses can be identified by means of immunohistochemical staining. However, this test is rather inconclusive because of its low sensitivity. Serological tests to detect HPV-specific antibodies in the serum of patients are of no importance for diagnostics because in only 50% of all cases can antibodies be detected, even in patients suffering from cervical cancer.
Methods used to detect viral nucleic acids in clinical tissue samples are very important for diagnostics. Particularly important are those methods that allow one to differentiate between individual types of a low-risk and high-risk HPV infection, since only the HPV types HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV66, HPV68, and HPV82 are associated with the development of carcinomas of the cervix in situ, and HPV53 probably is also carcinogenic.
One prior-art method for detecting HPV nucleic acids is the HCM (hybrid capture microplate) method from the Digene company (HC2 HPV DNA test), which is based on a signal-amplifying hybridization method. HPV-specific RNA sequences are used as the hybridization probes. After the probes have been incubated with denatured HPV-DNA from infected tissue, the RNA/DNA hybrids that are formed are captured on the surface of the microplate by means of specific antibodies. The RNA/DNA hybrids are detected by means of a second antibody that is marked with alkaline phosphatase. This enzyme can generate measurable light after the addition of certain substances. The HCM method permits subgroup-specific differentiation between low-risk types 6, 11, 42, 43, and 44 and high-risk types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68, and therefore can be useful in the differential diagnostics of unclear cytological findings. One disadvantage of this method, though, is that some high-risk types cannot be detected. Moreover, a cross-reaction occurs between the two subgroups.
Many methods for detecting viral nucleic acids in clinical tissue samples that are known in the prior art are based on prior HPV gene amplification. The PCR method has proved to be the most sensitive. By using such methods, a relatively broad spectrum of HPV viruses can be detected and then be typed. This typing is mainly accomplished by means of a sequence analysis of the PCR amplification product. The typing allows the HPV type to be described precisely, not only in the case of individual infections, but also in the case of multiple or mixed infections, so that it provides a means of assessing the oncogenic potential of the detected HPV genotypes that goes beyond cytological findings.
Snijders et al. J. Gen. Virol., 71 (1990), 173 to 181, and Surentheran et al., J. Clin. Path., 51 (1998), 606-610, describe the PCR process for detecting HPV-DNA. In this process, primer pairs that lie within HPV structure gene L1 are used. The amplified gene fragment is then sequenced in order to classify the detected HPV types. A disadvantage of the two methods, however, is that the L1 gene is less well preserved than other areas of the HPV-DNA. Therefore, only a limited number of HPV types can be detected using the methods described above. For example, the primers described by Snijder et al. can only detect some of the HPV types, such as HPV30, HPV39, and HPV51, with greatly reduced sensitivity. In addition, when the primers described by Snijder et al. are used, some HPV types, such as HPV18, result in the formation of additional bands.
DE 100 09 143 A1 describes the PCR process for detecting a general HPV infection, in particular, however, for detecting HPV-DNA in the anogenital area. In this process two primer pairs, which lie within the preserved HPV gene E1, are used. However, only the amplification products that are obtained using one of the primer pairs can be used for reliable typing, but not the amplification products obtained from the second primer pair. Another disadvantage is that the typing is carried out by sequencing the amplification products, or the amplification products have to be studied by means of temperature-gradient gel electrophoresis. Both sequencing and performing a temperature-gradient gel electrophoresis are labor- and cost-intensive procedures.
One significant disadvantage of the prior-art commercial tests for detecting and typing the HPV genotypes mainly is due to the fact that these tests only detect a limited spectrum of HPV types, and, in particular, rare genital HPV types are not adequately detected, even though some of these virus types are known to have a high oncogenic potential. Another disadvantage is that a time- and cost-intensive sequencing must always be performed after the amplification in order to be able to carry out the typing.